The carotenoid compounds of the present invention comprise natural pigments. It is known that carotenoids are useful additives in manufacturing of food products, feed products, cosmetics or pharmaceuticals.
The natural pigments of carotenoids are responsible for many of the yellow, orange, red or reddish colours seen in living organisms. In particular, astaxanthin is responsible for the red colour of crustaceae, molluscs and salmons, which cannot synthesize astaxanthin de novo and therefore it is necessary to add it to the diets of these animals.
Carotenoids are widely distributed in nature comprising plant and animal kingdoms. The colour varies dependent on the lengths of the chromophore and the type of the oxygen-containing groups attached. Carotenoid pigment formation is known from yeasts, certain bacteria, fungi and unicellular algae.
The carotenoids have in principle two biological functions. They serve as light-harvesting pigments in photosynthesis and they protect against photo oxidative damage caused by active oxygen species such as O2., H2O2 or OH., which are continuously generated in living cells.
Furthermore, carotenoids can absorb photons and transfer the energy to chlorophyll, thus assisting in the harvesting of sunlight.
It is further known that β-carotene, a precursor of astaxanthin, protects against radiation by absorption of energy in the blue region of the light spectrum. It is suggested that β-carotene in the body may protect against cancer and that it also functions as a precursor of vitamin A in mammals so that it is involved in provitamin A activity.
It has been shown that carotenoids, in particular astaxanthin, protect the skin from the damaging effects of ultraviolet radiation and ameliorate age-related macular degeneration. In addition, astaxanthin increases high density lipoproteins and protects against cardiovascular diseases.
The function of astaxanthin as a powerful antioxidant in animals is well known. Astaxanthin is a strong inhibitor of lipid peroxidation and has been shown to play an active role in the protection of biological membranes from oxidative injury. According to recent investigations it has been scrutinized that astaxanthin also shows chemo-preventive effects and reduce the incidence of chemically induced urinary bladder cancer in mice. In addition it has also been demonstrated that astaxanthin exerts immunomodulating effects by enhancing antibody production. These preliminary results suggest that astaxanthin could play an important role in cancer and tumor prevention, as well as eliciting a positive response from the immune system, thus making astaxanthin to a promising candidate in medicine and for the pharmaceutical industry.
Besides these physiological and medical functions, astaxanthin further plays or may play a role as antioxidant, hormone precursor, in reproduction, in growth and maturation.
Because of the powerful antioxidant activity of astaxanthin it is starting to be used in the human health-food sector. The antioxidant activities of astaxanthin have been shown to be approximately ten times greater than other carotenoids, such as zeaxanthin, lutein, cantaxanthin and β-carotene, and over 500 times greater than α-tocopherol, also known as vitamin E.
Astaxanthin is utilized mainly as nutritional supplement, which provides pigmentation in a wide variety of aquatic animals. In Far East it is used also for feeding poultry to yield a typical pigmentation of chicken. It is also a desirable and effective non-toxic colouring for the food industry and is valuable for cosmetics. It has also been shown that astaxanthin is a potent antioxidant in humans and thus is a desirable food additive.
Many researchers remark the vital role that especially astaxanthin plays in the physiology and in overall health, and suggest that astaxanthin is an essential nutrient that should be included in all aquatic diets at a minimum level of 5-10 parts per million (ppm).
While astaxanthin is a natural nutritional component, it can be found as a food supplement. The supplement is intended for human, animal, and aquaculture consumption. The commercial production of astaxanthin comes from both natural and synthetic sources. The FDA approved astaxanthin as a food colouring or colour additive for specific uses in animal and fish foods. The European Commission considers it as food dye within the E number system (E161j).
Therefore, Carotenoids, especially natural born astaxanthin, have a high industrial value as a safe natural food and feed additive, such as a colour improver, for fishes such as salmon, trout or red sea bream. In addition, it is promising as an additive in cosmetics and pharmaceuticals. Hence, there is an increasing interest in developing biological production of carotenoids, especially astaxanthin.
Until recently, the commercial interest focussed on the yeast Phaffia rhodozyma, but astaxanthin contents are low (0.3-0.5% dry weight) even after considerable strain improvement efforts. Shrimps shell waste is another potential source of astaxanthin, but direct use of the material is very low in astaxanthin (0.0025% dry weight). As a result, the quantities required in the feeds for efficient pigmentation add deleterious bulk and ash to the final feeds. The composition of astaxanthin esters in Haematococcus is similar to that of crustaceans, the natural dietary source of salmons. Moreover, all of the free astaxanthin and its esters in Haematococcus have 3S,3′S chirality, the same as in free salmonids, whereas Phaffia contains 3R,3′R astaxanthin and the synthetic one is a mixture of three isomers.
Currently, most of the astaxanthin is supplied, mainly for aquaculture, through chemical synthesis. However, because astaxanthin is a complex molecule and the synthesis is difficult. Therefore, the industrial use of carotenoids is hampered by the fact that synthetic carotenoid production, in particular the synthetic astaxanthin production, as well as the process for isolating natural astaxanthin are expensive, laborious and subject to seasonal variations.
A process for producing carotenoids, such methods as chemical synthesis, production by microorganisms, and extraction from natural products or sources are known in the art. As processes of chemical synthesis, the conversion of β-carotene and the synthesis from C15-phosphonium salts is also known for a long time.
As processes for producing β-carotene synthesis from β-ionone and extraction from green, yellow or red vegetables such as carrots, potatoes or pumpkins is known.
Carotenoids obtained by microorganisms, in particular astaxanthin obtained by microorganisms such as algae, have many advantages in comparison to the synthetic one, such as better retention in the fish gut, and a better acceptance by the consumers. In addition, regulations on the use of synthetic dyes in the food, cosmetic and pharmaceutical industry are currently very stringent. In this regard, Haematococcus algae meal has been approved in Japan, Europe and USA as a natural food colour and as a pigment for fish feeds.
The above-described production processes have various severe problems. Firstly, safety is not assured for the synthesized products; secondly, the production by microorganisms is low in productivity; thirdly, extraction from natural products or sources requires high costs. In particular, the latter is especially valid for astaxanthin since extraction from natural sources such as hill or crawfish requires high costs since the content of astaxanthin is extremely small and yet the extraction is difficult and time consuming. In addition, astaxanthin producing microorganisms are generally characterized in that they have a low growth rate, produce only small amounts of astaxanthin and have a robust cell wall that makes the extraction of the carotenoid difficult and thus not economical.
In particular, in the case of the green alga Haematococcus pluvialis the majority of these problems are combined. In addition to its extremely low growth rate, it is known that the cultures of this microorganism are easily contaminated.
In contrast to natural occurring astaxanthin the synthetic substance consists of a mixture of the (3S,3′S)-, (3S,3′R)- and (3R,3′R)-isomers and is commercially available under the trade name Carophyll® Pink.
Natural (3S,3′S) astaxanthin is limited in availability. Currently, in spite of the above-mentioned disadvantages and because of the lack of any alternatives it is commercially extracted from crustacea species and Haematococcus pluvialis. 
However, the success of commercial mass production of carotenoids, especially of astaxanthin by Haematococcus pluvialis, is hampered by a relatively low productivity of the cultures. This raises the production costs in such a way that in particular Haematococcus astaxanthin cannot compete on price against the synthetic pigment, which in turn consists of a mixture of isomers and laws or rules limit its commercial application as set forth supra.
There are several articles in the literature about mutants of Haematococcus pluvialis, but none of them describe any successful results as to the production of a carotenoid with high yield. For example, Tjahjono et al. used ethyl methansulfonate (EMS) to mutagenized Haematococcus pluvialis cells and three carotenoid biosynthesis inhibitors (norflurazon, fluridone and nicotine) for selection of resistant colonies. Some resistant mutants were obtained, but no one exhibited an enhanced volumetric content of carotenoids. The work consists in isolation of resistant mutants from the green alga Haematococcus pluvialis and their hybrid formation by protoplast fusion for breeding of higher astaxanthin producers. The fusion of protoplasts produces usually very unstable organisms that have to be kept in selective medium, in addition the growth of the mutants was much lower than the wild strains (Tjahjono, A. E. et al. J. Ferment. Bioeng. 77: 352-357, 1994).
In another article EMS and UV light were used for mutagenesis of Haematococcus pluvialis and compactin was used for selection of the mutants. Data of only two resistant mutants are presented, which have 1.4 and 2.0 fold higher astaxanthin content per cell than the wild type. However, the mutants grew slower and attained lower cell densities than the wild strain and no enhancement of the volumetric content could be measured in the mutant strains (Chumpolkulwong N. et al., Isolation and characterization of compactin resistant mutants of an astaxanthin synthesizing green alga Haematococcus pluvialis. Biotechnol. Lett. 19: 299-302, 1997).
In a more recent study that was published 6 to 9 years later from the former studies UV light or EMS induced mutagenesis has been used in two rounds and mutant selection on nicotine, diphenylamine, fluridone or norflurazon supplemented medium. The first round of mutagenesis gave rise to 1.6 and 1.7 times (w/w) more astaxanthin-rich mutants by screening with nicotine. In the second round of mutagenesis one of these two mutants was improved somewhat more to reach 2.1-fold increase in astaxanthin content in comparison with the wild strain. However, the growth rate of the mutants was much lower than that of the wild strain (Chen Y., et al. Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis, Biotech. Lett. 25: 299-302, 2003).
For the reasons mentioned above the known processes for industrial production of astaxanthin from algae, especially from Haematococcus pluvialis, seems to be impracticable or are at least very difficult and full of disadvantages. Moreover, the known processes are not attractive due to high costs, low contents in natural sources, laborious extraction processes with unsatisfying or disappointing results and lack of constant availability of the resources.
Hence, it is desirable to find a method or process for producing and providing carotenoid compounds or carotenoid pigments, a method for generating microorganisms producing high quantities of carotenoid pigments or cells containing high quantities of carotenoid pigments, respectively, as well as inexpensive means in order to make these carotenoids attractive and economical for industrial productivity and industrial application.
In particular, it is desirable to find an inexpensive, economical and non-laborious source for the production of (3S,3′S) astaxanthin for commercial or industrial and medical purposes
It is therefore an object of the present invention to provide an effective and economically advantageous method for producing a carotenoid, in particular to provide an effective and economically advantageous method for the production of astaxanthin.
It is a further object of the present invention to provide a carotenoid in isolated or purified or extracted or enriched form obtainable from or obtained by a microorganism capable of producing carotenoids with algae a high rate, preferably capable of producing astaxanthin with a high rate.
It is a further object of the present invention to provide a microorganism useful in the production of a carotenoid, preferably in the production astaxanthin, which is easily manageable, easily cultivable and from which the desired product can be obtained in high yields and economically.
It is an addition object of the present invention to provide products and goods containing a microorganism capable of producing carotenoids with a high rate, preferably capable of producing astaxanthin with a high rate.
It is an addition object of the present invention to provide products and goods containing a carotenoid or a carotenoid pigment in an isolated or purified or extracted or enriched form obtainable from or obtained by a microorganism capable of producing carotenoids with a high rate, preferably capable of producing astaxanthin with a high rate.
It is also an object of the present invention to provide the use of a microorganism capable of producing carotenoids economically and in high yields, preferably astaxanthin, for the manufacture of products and goods, especially for the production of animal feed, food, cosmetics or pharmaceuticals or additives thereto.
It is also a further object of the present invention to provide the use of a carotenoid, in particular astaxanthin, produced by a suitable microorganism for the manufacture of products and goods, especially for the production of animal feed, food, cosmetics or pharmaceuticals or additives thereto.
Furthermore it is an object of the present invention to provide the use of a carotenoid or a carotenoid pigment in an isolated or purified or extracted or enriched or immobilized form for the manufacture of products and goods, especially for the production of animal feed, food, cosmetics or pharmaceuticals or additives thereto, the carotenoid or carotenoid pigment is obtainable from or obtained by a microorganism capable of producing carotenoids with a high rate, preferably capable of producing astaxanthin with a high rate.
In addition it is an object of the present invention to provide a kit or a kit-of-parts comprising a carotenoid or a carotenoid pigment in isolated or purified or extracted or enriched or immobilized form or a suitable microorganism useful in the production of a carotenoid, preferably in the production astaxanthin, and/or products and goods containing a carotenoid, preferably astaxanthin, and/or a finished product or a manufactured article, which may or may not contain a carotenoid, preferably astaxanthin, which are contained or packaged spatially separated in one or more containers.
The biosynthesis as well as the production of carotenoids by different microorganisms is known in the art.
Examples of astaxanthin-producing microorganisms include the red yeast Phaffia rhodozyma, bacteria belonging to the Genera Brevibacterium, Mycobacterium and Agrobacterium, for example Agrobacterium alcaligenes, and the green alga Haematococcus pluvialis. 
It is well known that in prokaryotes conserved enzyme catalysed reactions mediate the early reactions of carotenoid biosynthesis which seem to follow the same route in all prokaryotic and eukaryotic organisms.
The de novo biosynthesis of carotenoids is starting from isoprenoid precursors, commonly beginning with acetyl-CoA, which is then converted to mevalonic acid.
The specific part of the pathway begins with the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, which is a colourless carotene, catalysed by prenyl transferases. A head-to-head condensation of two molecules of geranylgeranyl pyrophosphate leads to prephytoene pyrophosphate. In a subsequent two-step reaction the pyrophosphate moiety is removed and the colourless 15-cis-phytoene is formed.
Following four desaturation (also dehydrogenation) reactions 15-cis-phytoene is converted to lycopene. It should be noted that each of this membrane-bound dehydrogenation reactions increase the number of conjugated double bonds by two such that the number of conjugated double bonds increases from three in 15-cis-phytoene to eleven in lycopene, which is the pigment making the mature tomatoes red.
From cyanobacteria, algae and plants it is known that a single membrane-bound enzyme phytoene desaturase catalyse the first two desaturation reactions, from 15-cis-phytoene to ζ-carotene. Since the ζ-carotene is mostly in the all-trans configuration, a cis-trans isomerization is presumed at this site in the pathway. Again, in cyanobacteria, algae and plants ζ-carotene in transformed to lycopene via neurosporene.
Two cyclisation reactions are catalysed by a single membrane-bound enzyme lycopene β-cyclase converting lycopene to β-carotene.
The known xanthophyll variants are formed by the addition of various oxygen-containing side groups, such as hydroxy-, methoxy-, oxo-, epoxy-, aldehyde or carboxylic acid moieties. However, in the end little is known about the formation of xanthophylls. What is known is that hydroxylation of β-carotene requires molecular oxygen in a mixed-function oxidase reaction. The oxygenation and hydroxylation reactions leading to astaxanthin are catalysed by β-carotene oxygenase or β-carotene hydroxylase, respectively.
The lipophilic pigment astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione) was first described in aquatic crustaceans as an oxidized from of β-carotene. This pigment was later found to be very common in many marine animals and algae. However, only few animals can synthesize astaxanthin de novo from other carotenoids and most of them obtain it in their food. In the plant kingdom, astaxanthin occurs mainly in some species of cyanobacteria, algae and lichens.